Cells and Geneblazer® D1-CRE-Bla CHO-K1 Cells Contain

Version No.: GeneBLAzer ® Validation Packet Page 1 of 4 01Sep08

Optimization of the GeneBLAzer ® D1 CRE-bla CHO-K1 Cell Line

GeneBLAzer ® D1 CHO-K1 DA Assay Kit

GeneBLAzer ® D1 CRE-bla CHO-K1 Cells

Catalog Numbers – K1311 and K1709

Cell Line Descriptions GeneBLAzer ® D1 CHO-K1 DA (Division Arrested) cells and GeneBLAzer ® D1-CRE-bla CHO-K1 cells contain the human dopamine receptor 1 (D1) (Accession # NM_000794.2 ) stably integrated into the CellSensor ® CRE-bla CHO-K1 cell line. CellSensor ® CRE-bla CHO-K1 cells (Cat. no. K1535) contain a beta-lactamase (bla ) reporter gene under control of the Cyclic AMP Response Element (CRE). Division Arrested (DA) cells are available as an Assay Kit (which includes cells and sufficient substrate to analyze 1 x 384-well plate).

DA cells are irreversibly division arrested using a low-dose treatment of Mitomycin-C, and have no apparent toxicity or change in cellular signal transduction. Both GeneBLAzer ® D1 CHO-K1 DA cells and GeneBLAzer ®

D1-CRE-bla CHO-K1 cells are functionally validated for Z’-factor and EC 50 concentrations of dihydrexidine (Figure 1). In addition, GeneBLAzer ® D1-CRE-bla CHO-K1 cells have been tested for assay performance under variable conditions. Additional testing data using alternate stimuli are also included.

Target Description

Receptors for the neurotransmitter dopamine in the brain have long been targets for anti-psychotic and Parkinson’s disease drugs (1-4). The dopamine family of GPCR’s consists of 5 members that are separated into two groups based on sequence homology: D1-like and D2-like. The D1-like receptors are D1 and D5 (5, 6), while D2, D3 (7), and D4 (8) make up the D2-like group. Activaton of the D1-like receptors (D1 and D5) is associated with activation of adenylate cyclase whereas activation of D2-like receptors (D2, D3, and D4) is associated with inhibition of adenylate cyclase activity.

Have a question? Contact our Technical Support Team NA: 800-955-6288 or INTL: 760-603-7200 Select option 3, ext. 40266 Email: [email protected] Optimization of the GeneBLAzer ® D1 CRE-bla CHO-K1 Cell Line Page 2 of 4

Validation Summary Primary Agonist Dose Response

Testing and validation of this assay was evaluated Figure 1 — GeneBLAzer ® D1 CHO-K1 DA and D1-CRE-bla in a 384-well format using LiveBLAzer™-FRET B/G CHO-K1 dose response to dihydrexidine under optimized conditions Substrate. 110 Dividing Cells 1. Dihydrexidine agonist dose response 100 90 DA Cells under optimized conditions 80 70 60 DA cells Dividing Cells 50 EC 50 71 nM 77 nM 40 Z’-factor 0.92 0.89 30 % Activation 20 10 Recommended cell no. = 10K cells/well 0 Recommended [DMSO] = 0.1-1% -10 -12 -11 -10 -9 -8 -7 -6 -5 -4 Recommended Stim. Time = 5 hours Log [Dihydrexidine] M Max. [Stimulation] = 35 µM

® ® 2. Alternate agonist dose response GeneBLAzer D1 CHO-K1 DA cells and GeneBLAzer D1-CRE - bla SKF38393 EC = 22 nM CHO-K1 cells (10,000 cells/well) were plated in a 384-well 50 format and incubated for 16-20 hours. Cells were stimulated Fenoldopam EC = 32 nM 50 with a dilution series of dihydrexidine (Phoenix Pharmaceutical SKF75670 EC 50 = 86 nM #019-06) in the presence of 0.5% DMSO for 5 hours. Cells Dopamine EC 50 = 454 nM were then loaded with LiveBLAzer™-FRET B/G Substrate for 2 hours. Fluorescence emission values at 460 nm and 530 nm were obtained using a standard fluorescence plate reader and 3. Antagonist Dose Response % Activation plotted for each replicate against the concentrations of dihydrexidine (n=6 for each data point). SCH23390 IC = 42 nM 50 SKF-83566 IC = 89 nM 50

4. Agonist 2 nd Messenger Response Alternate Agonist Dose Response

Dihydrexidine EC = 47 nM 50 Figure 2 — GeneBLAzer ® D1-CRE-bla CHO-K1 dose response to alternate agonists under optimized conditions

120 110 Dihydrexidine 100 SKF 75670 hydrobromide 90 80 Fenoldopam 70 SKF-38393 hydrochloride 60 50 Dopamine Hydrochloride 40 30 % Activation 20 10 0 -10 -10 -9 -8 -7 -6 -5 -4 Log [Agonist] M

GeneBLAzer ® D1-CRE-bla CHO-K1 cells (10,000 cells/well) were plated the day before the assay in a 384-well format and stimulated with Dihydrexidine, SKF-75670, Fenoldopam, SKF- 38393, or Dopamine over the indicated concentration range in the presence of 0.1% DMSO for 5 hours. Cells were then loaded with LiveBLAzer™-FRET B/G Substrate for 2 hours. Fluorescence emission values at 460 nm and 530 nm were obtained using a standard fluorescence plate reader and the % Activation plotted against the indicated concentrations of agonist (n=2 for each data point).

Antagonist Dose Response Agonist 2 nd Messenger Dose Response

Figure 3— GeneBLAzer ® D1-CRE-bla CHO-k1 antagonist Figure 4— GeneBLAzer ® D1-CRE-bla CHO-k1 2 nd dose response under optimized conditions messenger dose response to dihydrexidine under optimized conditions.

110 110 100 (+) SCH23390 100 90 (-) SKF-83566 90 80 80 70 70 60 60 50 50 40 40 30 30 % Inhibition 20 % Activation 20 10 10 0 0 -10 -10 -12 -11 -10 -9 -8 -7 -6 -5 -4 -10 -9 -8 -7 -6 -5 -4 Log [Antagonist] M Log [Dihydrexidine] M ® bla GeneBLAzer D1-CRE- CHO-K1 cells (10,000 cells/well) GeneBLAzer ® D1-CRE-bla CHO-K1 cells were tested for a were plated in a 384-well assay plate and incubated for 16-20 response to dihydrexidine (Phoenix Pharmaceutical #019-06) hours. Cells were then incubated with a dilution series of with a TR-FRET cAMP assay antagonist for 30 min. at 37°C followed by a 5 hour incubation with an EC 80 concentration of dihydrexidine (Phoenix Pharmaceutical #019-06) in 0.1% DMSO. Fluorescence emission values at 460 nm and 530 nm were obtained using a standard fluorescence plate reader and the % Inhibition plotted against the indicated concentrations of antagonist.

References

1. Carlsson, A., Lindqvist, M. and Magnusson, M. (1957) 3,4-Dihydroxyphenylalanine and 5- hydroxytryptophan as reserpine antagonists. Nature , 180 , 1200.

2. Carlsson, A., Lindqvist, M., Magnusson, T. and Walbeck, B. (1958) On the presence of 3- hydroxytyramine in brain. Science , 127 , 471 .

3. Carlsson, A. and Lindqvist, M. (1963) Effect of chlorpromazine or haloperidol on formation of 3-methoxytyramine and normetanephrine in mouse brain . Acta Pharmacol. Toxicol ., 20 , 140 - 144.

4. Moran, T. H., Robinson, P. H., Goldrich, M. S. and McHugh, P. R. (1963) Die topishe Lokalisation und das Verhalten von Noradrenalin und Dopamin (3-hydroxytyramin) in der Substantia nigra des normalen und Parkinson-kranken Menschen. Wien. Klin. Wochenschr ., 75 , 309 - 312.

5. Van Tol, H. H. M. et al. (1992) Multiple dopamine D4receptor variants in the human population . Nature , 358 , 149 - 152.

6. Sunahara, R. K. et al. (1991) Cloning of the gene for a human dopamine D5 receptor with higher affinity for dopamine than D1. Nature , 350 , 614 - 619.

7. Sokoloff, P., Giros, B., Martres, M.-P., Bouthenet, M.-L. and Schwartz, J.-C. (1990) Molecular cloning and characterization of a novel dopamine receptor (D3) as a target for neuroleptics. Nature , 347 , 146 - 151.

8. Van Tol, H. H. M., Bunzow, J. R., Guan, H. C., Sunahara, R. K., Seeman, P., Niznik, H. B. and Civelli, O. (1991) Cloning of the gene for a human dopamine D4receptor with high affinity for the antipsychotic clozapine. Nature , 350 , 610 - 614.

Have a question? Contact our Technical Support Team NA: 800-955-6288 or INTL: 760-603-7200 Select option 3, ext. 40266 Email: [email protected]